Virtual Absence Of Ca2 Research Articles

Insights into the release mechanism of astrocytic glutamate evoking in neurons NMDA receptor-mediated slow depolarizing inward currents.

The gliotransmitter glutamate in different brain regions modulates neuronal excitability and synaptic transmission through a variety of mechanisms. Among the hallmarks of astrocytic glutamate release are the slow depolarizing inward currents (SICs) in neurons mediated by N-methyl-d-aspartate receptor activation. Different stimuli that evoke Ca2+ elevations in astrocytes induce neuronal SICs suggesting a Ca2+ -dependent exocytotic glutamate release mechanism of SIC generation. To gain new insights into this mechanism, we investigated the relationship between astrocytic Ca2+ elevations and neuronal SICs in mouse hippocampal slice preparations. Here we provide evidence that SICs, occurring either spontaneously or following a hypotonic challenge, are unchanged in the virtual absence of Ca2+ signal changes at astrocytic soma and processes, including spatially restricted Ca2+ microdomains. SICs are also unchanged in the presence of Bafilomycin A1 that after prolonged slice incubation depletes glutamate from astrocytic vesicles. We also found that hemichannels and TREK family channels-that recent studies proposed to mediate astrocytic glutamate release - are not involved in SIC generation. SICs are reduced by the volume-sensitive anion channel antagonists diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), quinine and fluoxetine, suggesting a possible contribution of these channels in SIC generation. Direct measurements of astrocytic glutamate release further confirm that hypotonicity-evoked gliotransmission is impaired following DIDS, quinine and fluoxetine while the exocytotic release of glutamate-that is proposed to mediate synaptic transmission modulation by astrocytes-remains unaffected. In conclusion, our data provide evidence that the release of glutamate generating SICs occurs independently on exocytotic Ca2+ -dependent glutamate release mechanism.

PC-PLC/sphingomyelin synthase activity plays a central role in the development of myogenic tone in murine resistance arteries.

Myogenic tone is an intrinsic property of the vasculature that contributes to blood pressure control and tissue perfusion. Earlier investigations assigned a key role in myogenic tone to phospholipase C (PLC) and its products, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). Here, we used the PLC inhibitor, U-73122, and two other, specific inhibitors of PLC subtypes (PI-PLC and PC-PLC) to delineate the role of PLC in myogenic tone of pressurized murine mesenteric arteries. U-73122 inhibited depolarization-induced contractions (high external K(+) concentration), thus confirming reports of nonspecific actions of U-73122 and its limited utility for studies of myogenic tone. Edelfosine, a specific inhibitor of PI-PLC, did not affect depolarization-induced contractions but modulated myogenic tone. Because PI-PLC produces IP3, we investigated the effect of blocking IP3 receptor-mediated Ca(2+) release on myogenic tone. Incubation of arteries with xestospongin C did not affect tone, consistent with the virtual absence of Ca(2+) waves in arteries with myogenic tone. D-609, an inhibitor of PC-PLC and sphingomyelin synthase, strongly inhibited myogenic tone and had no effect on depolarization-induced contraction. D-609 appeared to act by lowering cytoplasmic Ca(2+) concentration to levels below those that activate contraction. Importantly, incubation of pressurized arteries with a membrane-permeable analog of DAG induced vasoconstriction. The results therefore mandate a reexamination of the signaling pathways activated by the Bayliss mechanism. Our results suggest that PI-PLC and IP3 are not required in maintaining myogenic tone, but DAG, produced by PC-PLC and/or SM synthase, is likely through multiple mechanisms to increase Ca(2+) entry and promote vasoconstriction.

Spontaneous oscillatory contraction (SPOC) in cardiomyocytes.

SPOC (spontaneous oscillatory contraction) is a characteristic state of the contractile system of striated (skeletal and cardiac) muscle that exists between the states of relaxation and contraction. For example, Ca-SPOCs occur at physiological Ca2+ levels (pCa ∼6.0), whereas ADP-SPOC occurs in the virtual absence of Ca2+ (pCa ≥ 8; relaxing conditions in the presence of MgATP), butin the presence of inorganic phosphate (Pi) and a high concentration of MgADP. The concentration of Mg-ADP necessary for SPOC is nearly equal to or greater than the MgATP concentration for cardiac muscle and is several times higher for skeletal muscle. Thus, the cellular conditions for SPOC are broader in cardiac muscle than in skeletal muscle. During these SPOCs, each sarcomere in a myofibril undergoes length oscillation that has a saw-tooth waveform consisting of a rapid lengthening and a slow shortening phase. The lengthening phase of one half of a sarcomere is transmitted to the adjacent half of the sarcomere successively, forming a propagating wave (termed a SPOC wave). The SPOC waves are synchronized across the cardiomyocytes resulting in a visible wave of successive contractions and relaxations termed the SPOC wave. Experimentally, the SPOC period (and therefore the velocity of SPOC wave) is observed in demembranated cardiomyocytes and can be prepared from a wide range of animal hearts. These periods correlate well with the resting heartbeats of a wide range of mammals (rat, rabbit, dog, pig and cow). Preliminary experiments showed that the SPOC properties of human cardiomyocytes are similar to the heartbeat of a large dog or a pig. This correlation suggests that SPOCs may play a fundamental role in the heart. Here, we briefly summarize a range of SPOC parameters obtained experimentally, and relate them to a theoretical model to explain those characteristics. Finally, we discuss the possible significance of these SPOC properties in each and everyheartbeat.

Coupled gating modifies the regulation of cardiac ryanodine receptors by luminal Ca2 +

Cardiac ryanodine receptors (RYR2s) infrequently exhibit coupled gating that is manifested by synchronous opening and closing. To better characterize this phenomenon, we investigated the regulation of coupled RYR2 channels by luminal Ca2+ focusing on effects that are likely mediated by the true luminal activation mechanism. By reconstituting an ion channel into a planar lipid bilayer and using substantially lower concentration of luminal Ba2+ (8mM, the virtual absence of Ca2+) and luminal Ca2+ (8mM), we show that response of coupled RYR2 channels to caffeine at a diastolic cytosolic Ca2+ (90nM) was affected by luminal Ca2+ in a similar manner as for the single RYR2 channel except the gating behavior. Whereas, the single RYR2 channel responded to luminal Ca2+ by prolongation in open and closed times, coupled RYR2 channels seemed to be resistant in this respect. In summary, we conclude that the class of Ca2+ sites located on the luminal face of coupled RYR2 channels that is responsible for the channel potentiation by luminal Ca2+ is functional and not structurally hindered by the channel coupling. Thus, the idea about non-functional luminal Ca2+ sites as a source of the apparent gating resistance of coupled RYR2 channels to luminal Ca2+ appears to be ruled out.

The Nature of Ca2+-Controlled Gating in the Human Cardiac Ryanodine Receptor (hRyR2): A Zero Ca2+ Approach

RyR2-mediated intracellular Ca2+ release is pivotal in maintaining the rhythmic contraction of cardiac myocytes. The precise mechanism by which binding of cytosolic Ca2+ triggers gating is unknown. Functional studies of single RyR2s in lipid bilayers provide insights into gating behaviour as detailed structural information regarding the nature of the Ca2+-dependent gate or Ca2+ binding site(s) is as yet unavailable.This study aims to uncover the properties of the Ca2+-controlled ‘gate' by examining changes in single channel gating in the virtual absence of Ca2+. To visualize these changes, ryanodine was employed to keep the channel open in a subconductance state in the presence and absence of Ca2+, while K+ was used as the permeant ion for better resolution of single channel currents. On reducing the Ca2+ from contaminant levels to nominally zero on both cytosolic and luminal sides of the ryanodine-modified hRyR2 (using 3.5mM EGTA), a significant decrease in channel Po is seen at positive (current direction: cytosolic-luminal) holding potentials but not in the physiological direction of current flow (0.99±0.002 to 0.75±0.05 at +40mV (p<0.0005) and 0.99 to 0.98±0.009 at −40mV; n=9). Analysis of dwell times demonstrates that Po decreases by increasing the frequency of closings (∼170 fold increase at +40mV, ∼8-fold at −40mV). Further examination of the voltage sensitivity of the modified channel reveals a sharp dependence of activity on potential. High cytosolic Ba2+ had a direct inhibitory effect on the gating kinetics induced by the removal of Ca2+. This study demonstrates that ryanodine modifies RyR2 by acting on a Ca2+-controlled gate and gating of the modified channel is voltage-sensitive. HMM-based modeling of this data provides further insights into this fundamental process of Ca2+-induced channel opening. This study is supported by the BHF.

The beta1 subunit enhances oxidative regulation of large-conductance calcium-activated K+ channels.

Oxidative stress may alter the functions of many proteins including the Slo1 large conductance calcium-activated potassium channel (BKCa). Previous results demonstrated that in the virtual absence of Ca2+, the oxidant chloramine-T (Ch-T), without the involvement of cysteine oxidation, increases the open probability and slows the deactivation of BKCa channels formed by human Slo1 (hSlo1) α subunits alone. Because native BKCa channel complexes may include the auxiliary subunit β1, we investigated whether β1 influences the oxidative regulation of hSlo1. Oxidation by Ch-T with β1 present shifted the half-activation voltage much further in the hyperpolarizing direction (−75 mV) as compared with that with α alone (−30 mV). This shift was eliminated in the presence of high [Ca2+]i, but the increase in open probability in the virtual absence of Ca2+ remained significant at physiologically relevant voltages. Furthermore, the slowing of channel deactivation after oxidation was even more dramatic in the presence of β1. Oxidation of cysteine and methionine residues within β1 was not involved in these potentiated effects because expression of mutant β1 subunits lacking cysteine or methionine residues produced results similar to those with wild-type β1. Unlike the results with α alone, oxidation by Ch-T caused a significant acceleration of channel activation only when β1 was present. The β1 M177 mutation disrupted normal channel activation and prevented the Ch-T–induced acceleration of activation. Overall, the functional effects of oxidation of the hSlo1 pore-forming α subunit are greatly amplified by the presence of β1, which leads to the additional increase in channel open probability and the slowing of deactivation. Furthermore, M177 within β1 is a critical structural determinant of channel activation and oxidative sensitivity. Together, the oxidized BKCa channel complex with β1 has a considerable chance of being open within the physiological voltage range even at low [Ca2+]i.

Oxidative regulation of large conductance calcium-activated potassium channels.

Reactive oxygen/nitrogen species are readily generated in vivo, playing roles in many physiological and pathological conditions, such as Alzheimer's disease and Parkinson's disease, by oxidatively modifying various proteins. Previous studies indicate that large conductance Ca(2+)-activated K(+) channels (BK(Ca) or Slo) are subject to redox regulation. However, conflicting results exist whether oxidation increases or decreases the channel activity. We used chloramine-T, which preferentially oxidizes methionine, to examine the functional consequences of methionine oxidation in the cloned human Slo (hSlo) channel expressed in mammalian cells. In the virtual absence of Ca(2+), the oxidant shifted the steady-state macroscopic conductance to a more negative direction and slowed deactivation. The results obtained suggest that oxidation enhances specific voltage-dependent opening transitions and slows the rate-limiting closing transition. Enhancement of the hSlo activity was partially reversed by the enzyme peptide methionine sulfoxide reductase, suggesting that the upregulation is mediated by methionine oxidation. In contrast, hydrogen peroxide and cysteine-specific reagents, DTNB, MTSEA, and PCMB, decreased the channel activity. Chloramine-T was much less effective when concurrently applied with the K(+) channel blocker TEA, which is consistent with the possibility that the target methionine lies within the channel pore. Regulation of the Slo channel by methionine oxidation may represent an important link between cellular electrical excitability and metabolism.

Allosteric voltage gating of potassium channels I. Mslo ionic currents in the absence of Ca(2+).

Activation of large conductance Ca(2+)-activated K(+) channels is controlled by both cytoplasmic Ca(2+) and membrane potential. To study the mechanism of voltage-dependent gating, we examined mSlo Ca(2+)-activated K(+) currents in excised macropatches from Xenopus oocytes in the virtual absence of Ca(2+) (<1 nM). In response to a voltage step, I(K) activates with an exponential time course, following a brief delay. The delay suggests that rapid transitions precede channel opening. The later exponential time course suggests that activation also involves a slower rate-limiting step. However, the time constant of I(K) relaxation [tau(I(K))] exhibits a complex voltage dependence that is inconsistent with models that contain a single rate limiting step. tau(I(K)) increases weakly with voltage from -500 to -20 mV, with an equivalent charge (z) of only 0.14 e, and displays a stronger voltage dependence from +30 to +140 mV (z = 0.49 e), which then decreases from +180 to +240 mV (z = -0.29 e). Similarly, the steady state G(K)-V relationship exhibits a maximum voltage dependence (z = 2 e) from 0 to +100 mV, and is weakly voltage dependent (z congruent with 0.4 e) at more negative voltages, where P(o) = 10(-5)-10(-6). These results can be understood in terms of a gating scheme where a central transition between a closed and an open conformation is allosterically regulated by the state of four independent and identical voltage sensors. In the absence of Ca(2+), this allosteric mechanism results in a gating scheme with five closed (C) and five open (O) states, where the majority of the channel's voltage dependence results from rapid C-C and O-O transitions, whereas the C-O transitions are rate limiting and weakly voltage dependent. These conclusions not only provide a framework for interpreting studies of large conductance Ca(2+)-activated K(+) channel voltage gating, but also have important implications for understanding the mechanism of Ca(2+) sensitivity.

Arachidonic acid and diacylglycerol release associated with inhibition of myosin light chain dephosphorylation in rabbit smooth muscle.

1. Exogenous arachidonic acid (AA) inhibits the protein phosphatase that dephosphorylates smooth muscle myosin, thus sensitizing the contractile response to Ca2+; it also inhibits voltage-gated Ca2+ channels in smooth muscle. The purpose of the present study was to determine whether endogenous AA is increased by agonists in a manner consistent with its role as a messenger regulating myosin phosphatase and Ca2+ channels. Both AA and diacylglycerol (DAG) were measured in [3H]AA-labelled intact and permeabilized (with staphylococcal alpha-toxin) rabbit femoral arteries stimulated with the alpha 1-adrenergic agonist phenylephrine (PE) (intact and permeabilized smooth muscles) or by guanosine-5'-O-(3-thiotriphosphate (GTP gamma S; permeabilized smooth muscles in which the [Ca2+] was maintained constant). Arachidonic acid mass was determined with gas chromatography and mass spectrometry (GC-MS). 2. In intact smooth muscle, PE increased both AA and DAG levels significantly, to 210 and 145% of baseline values, respectively. Another Ca2+-sensitizing agent, the thromboxane analogue U46619, caused a similar increase in AA and DAG levels in rabbit pulmonary artery. 3. In permeabilized smooth muscle at constant [Ca2+](pCa 6.5) GTP gamma S-induced AA and DAG release preceded force development and GTP gamma S (50 microM, 10 min) increased AA mass to 61-88 microM. 4. Phorbol-12,13-dibutyrate (PDBu), another Ca2+-sensitizing agent, also increased both AA and DAG levels in permeabilized smooth muscle at pCa 6.5, whereas the inactive analogue, 4 alpha-phorbol, did not have a Ca2+-sensitizing effect, nor did it increase AA and DAG levels. 5. In the virtual absence of Ca2+ (pCa > 8) GTP gamma S also increased AA and DAG levels by 3.5- and 1.6-fold, respectively. The effect of free Ca2+ itself on AA and DAG release was modest in the physiological range (pCa 7.0 to pCa 6.0), but pCa 4.5 caused an approximately 3- to 4-fold increase in AA and DAG levels, compared with the levels at pCa 8. In permeabilized ileum smooth muscle maintained at constant [Ca2+] (pCa 6.0), carbachol also significantly increased AA to 1.75 times its original value within 1 min of its application. 6. Our results are consistent with, although do not prove, the roles of AA and DAG as second and/or co-messenger(s) in smooth muscle, while the increases in AA and DAG levels induced by PDBu raise the possibility that they contribute to some of the cellular effects of phorbol esters.

Ca(2+)- and GTP[gamma S]-induced translocation of the glucose transporter, GLUT-4, to the plasma membrane of permeabilized cardiomyocytes determined using a novel immunoprecipitation method.

In cardiomyocytes glucose transport is activated not only by insulin but also by contractile activity that causes translocation of the glucose transporter, GLUT-4, from intracellular vesicles to the plasma membrane. The latter effect may possibly be mediated by intracellular Ca2+, as suggested by previous studies. To investigate the role of Ca2+, we permeabilized neonatal rat myocytes with alpha-toxin and incubated them for 1 h either at a pCa (i.e.--log10 [Ca2+]) of 8 (control) or at a pCa of 5 in the presence of adenosine 5'-triphosphate (ATP). Translocation of GLUT-4 was then monitored by a novel immunoprecipitation method using a peptide antibody directed against an exofacial (extracellular) loop of GLUT-4 (residues 58-80). Incorporation of GLUT-4 into the plasmalemma was stimulated 1.8-fold by 10 microM Ca2+ and 1.7-fold by insulin (as in the case of intact cells). The insulin effect was Ca2+ independent, i.e. it was identical in the absence and presence of Ca2+ (10 microM). Guanosine 5'-O-(3-thio-triphosphate) (GTP[gamma S]), which was inactive in intact cells, also caused translocation of GLUT-4 in permeabilized cardiomyocytes. Thus, incorporation of GLUT-4 into the plasma membrane was enhanced 2.5-fold by 200 microM GTP[gamma S] in the virtual absence of Ca2+ (pCa 8) and even 3.5-fold at 10 microM free Ca2+. We conclude that an increase in intracellular Ca2+ concentration increases GLUT-4 translocation of (permeabilized) cardiomyocytes to a similar extent as do insulin and GTP[gamma S] in the absence of Ca2+, but that the effects of Ca2+ and GTP[gamma S] may be additive.

Extracellular somatostatin measured by microdialysis in the hippocampus of freely moving rats: evidence for neuronal release.

Intracerebral microdialysis combined with a sensitive and specific radioimmunoassay was used to monitor the neuronal release of somatostatin (somatostatin-like immunoreactivity, SLI) in the dorsal hippocampus of freely moving rats. The sensitivity of the radioimmunoassay was optimized to detect < 1 fmol/ml. The basal concentration of SLI in 20-min dialysate fractions (5 microliters/min) collected 24 h after probe implantation was stable over at least 200 min. The spontaneous efflux dropped by 54 +/- 6.4% (p < 0.05) when Ca2+ was omitted and 1 mM EGTA added to the Krebs-Ringer solution and by 65.5 +/- 3.2% (p < 0.05) in the presence of 1 microM tetrodotoxin. Depolarizing concentrations of the Na+ channel opener veratridine (6.25, 25, 100 microM) induced 11 +/- 2 (p < 0.05), 17 +/- 2 (p < 0.05), and 21 +/- 5 (p < 0.01) fold increase in SLI concentration, respectively, during the first 20 min of perfusion. The effect of 100 microM veratridine was blocked by coperfusion with 5 microM tetrodotoxin (p < 0.01) and reduced by 79% (p < 0.01) in the virtual absence of Ca2+. Neuronal depolarization by 20 min of perfusion with Krebs-Ringer solution containing 25 and 50 mM KCl and proportionally lowered Na+ increased the dialysate SLI 4.4 +/- 1 (p < 0.05) and 17 +/- 3 (p < 0.01) fold baseline, respectively. Ten micromolar ouabain, a blocker of Na+,K(+)-ATPase, increased the dialysate SLI 15-fold baseline, on average (p < 0.05), during 80 min of perfusion. The results demonstrate the suitability of brain microdialysis for monitoring the neuronal release of SLI and for studying its role in synaptic transmission.

Mechanisms of luteinizing-hormone exocytosis in Staphylococcus aureus-α-toxin-permeabilized sheep gonadotropes

We have used primary gonadotropes permeabilized with the pore-forming protein Staphylococcus aureus alpha-toxin to investigate luteinizing hormone (lutropin, LH) exocytosis. The diameter of the alpha-toxin pores (2-3 nm) allows the exchange of small molecules, whereas larger cytosolic proteins are retained. Because of the slow exchange of small molecules through the pores, we have developed a protocol which combines prolonged pre-equilibration of the permeabilized cells at 0 degrees C before stimulation with strong Ca2+ buffering. Under these conditions, increasing the free Ca2+ concentration from 0.1 microM to 10 microM [EC50 (concentration effecting half-maximal response) 2-3 microM] resulted in a 15-20-fold increase in LH exocytosis. LH exocytosis was maximal in the first 3 min and completed by 12 min. When permeabilized cells were equilibrated for prolonged periods in the absence of MgATP, Ca2(+)-stimulated LH secretion gradually declined (greater than 90% decrease by 60 min). Addition of MgATP (5 mM) rapidly restored full Ca2(+)-stimulated LH secretion. MgATP supported Ca2(+)-stimulated LH secretion at a half-maximal concentration of 1.5 mM. UTP and adenosine 5'-[gamma-thio]triphosphate were 40 and 31% as effective as MgATP, whereas other nucleotide triphosphates were ineffective. The protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA; 50 nM) stimulated LH exocytosis at free Ca2+ concentrations as low as 1 nM and was additive with Ca2+ at higher free Ca2+ concentrations. PMA-stimulated exocytosis required MgATP at concentrations similar to those required for Ca2(+)-stimulated LH exocytosis. These results demonstrate that LH exocytosis can be triggered both by micromolar Ca2+ concentrations or, in the virtual absence of Ca2+, by PKC activation. Both mechanisms of stimulated exocytosis have an absolute requirement for millimolar ATP. Because they retain cytosolic proteins, alpha-toxin-permeabilized cells may have advantages over alternative permeabilization methods provided that conditions are used that compensate for slow diffusion through alpha-toxin pores.

Characterization of increased Ca2+ efflux by quercetin from the sarcoplasmic reticulum in frog skinned skeletal muscle fibres.

To clarify further the characteristics of the Ca-releasing action of quercetin, we examined the effect of quercetin on Ca2+ efflux from the sarcoplasmic reticulum without Ca uptake activity by omission of ATP in the presence of a high concentration (10 mM EGTA) of Ca2+ buffer, using mechanically skinned fibres from frog skeletal muscles. Quercetin increases the rate of Ca release in the presence of Ca2+ and shifts the relationship between Ca2+ concentration and the rate of Ca release to a lower range of Ca2+ concentrations. AMP potentiates the effect of quercetin. Mg2+ and procaine decrease the rate of Ca release in the presence of quercetin. These findings indicate that quercetin is very similar to caffeine; in fact, experiments confirmed that quercetin shares the site(s) of action with caffeine. Subsequent findings, however, suggest that the mechanism of the drug-induced Ca release is not as simple as expected with caffeine which may increase the affinity for Ca2+ of the 'Ca2+-induced Ca-release' mechanism, and that drugs, Ca2+ and nucleotide interact with one another in a more complex way to open the gate for Ca release. In the presence of 10 mM EGTA quercetin also causes Ca release in the virtual absence of Ca2+ if a sufficient amount of AMP is present, and the rate of Ca release in the presence of 1.55 micron Ca2+ showed different dependence on AMP concentration with and without quercetin. A higher concentration of Mg2+ is required in the presence of AMP than in the absence of AMP in order to eliminate Ca release by quercetin.

A mutant of Chlamydomonas reinhardtii that lacks the flagellar outer dynein arm but can swim.

A new type of Chlamydomonas mutant, which lacks the outer dynein arm but can swim, was isolated. Sodium dodecyl sulphate/polyacrylamide gel electrophoresis showed that four of the ten high-molecular-weight bands of dynein present in the wild-type axoneme are missing or diminished in the mutant axoneme. The mutant has a swimming rate of about 35 micrometers/s and a flagellar beat frequency of about 25 Hz, both of which are about 1/2.5 to 1/3 of those of the wild type. The mutant flagella beat with an asymmetric, cilia-type pattern, similar to the forward-swimming mode of the flagellar beating pattern of the wild type. However, unlike wild-type flagella, the mutant flagella never beat with a symmetrical waveform: when the cells were stimulated by intense light, the mutant transiently stopped beating its flagella, whereas the wild-type cell transiently swam backwards with the two flagella beating with a symmetrical waveform. Both wild-type and mutant cells could be demembranated by Nonidet P40 and their swimming reactivated by addition of Mg-ATP in the virtual absence of Ca2+. Double reciprocal plots of the beat frequency against ATP concentrations showed a linear relationship for both strains, yielding maximal frequencies of 44 Hz (wild-type) and 23 Hz (mutant). The mutant axonemes can be reactivated only when the Ca2+ concentration is lower than 10(-6) M: at pCa4, the wild-type axonemes beat with a symmetrical waveform, but the mutant axonemes showed no movement. These findings indicate that the outer dynein arm is dispensable for flagellar beating of the asymmetric waveform (forward-swimming mode), but not for beating of the symmetrical waveform (backward-swimming mode), and thus suggest the importance of the outer dynein arm in the switching of flagellar waveforms.

The effect of sodium, calcium and metabolic inhibitors on calcium efflux from goldfish heart ventricles.

1. 45Ca efflux and tissue Ca content were examined in goldfish ventricles under conditions known to affect cellular Ca movements. 2. EGTA or Ca-EGTA was added to the washout solutions in sufficient concentration (10 mM) to avoid retardation of the apparent tissue 45Ca efflux by extracellular 45Ca binding or backflux. 3. After a variable initial increase, the cellular Ca content usually stabilizes within 60 min when ventricles are immersed in Li- or K-substituted saline containing 1.8 mM Ca0 (under these conditions the internal Ca2+ concentration is below 10(-5) M). 4. 45Ca efflux is maximally activated by external concentrations of Ca2+ as low as 10(-6) M, in both Na-containing and Na-free saline. 5. 45Ca efflux decreases in Na-free solutions. It is reactivated by Na-saline. The effect of different external Na concentration on 45Ca efflux is comparable at external Ca2+ concentrations between 10(-6) M and 2 x 10(-3) M. 6. Reactivation of Ca efflux after Na0 readmission is inhibited by metabolic poisoning, or in the presence of 10 mM-caffeine. Loading with 45Ca at very low external Ca2+ concentration prevents the inhibition of Ca efflux in Na-free medium. 7. Caffeine (10 mM) produces contractions of about equla size when K-depolarized preparations are immersed in either Na- or Li-saline. At the same time there is a similar increase in 45Ca efflux in absence of Na0 and in its presence. 8. In the virtual absence of Ca2+0 (10(-5) M-Ca, 10(-2) M-EGTA) and Na+0, the residual 45Ca efflux is reversibly inhibited by cyanide (2 mM). 9. The results are roughly compatible with the general concept of ATP-dependent Na-Ca exchange in internal Ca2+ homeostasis. However, this hypothesis should probably be modified to account for the fact that under physiological concentrations Na+0 and Ca2+0 do not compete for activating 45Ca efflux. Metabolic products may be involved in Na0- and Ca0-dependent Ca efflux. It is therefore not excluded that a Na-independent active mechanism co-operates with Na-Ca exchange in Ca extrusion.

Contraction of detergent-treated smooth muscle.

After exposure of segments of rabbit taenia coli to the nonionic detergent Triton X-100, tension could be induced by increasing the [Ca2+] in the micromolar range. In the presence of a saturating [Ca2+], this preparation developed nearly 100% of the control tetanus tension recorded from the intact muscle prior to the detergent treatment. In addition, tension could be induced by increasing the [Mg2+], in the virtual absence of Ca2+. Mg2+ seems to inhibit the Ca2+-induced tension in a predominantly competitive manner.